ProjectSummary/Abstract Thelong?termgoalsofthisprojectaretodevelopahigh?resolutionunderstandingofionchannelfunctionand regulation. Our studies focus on uncovering the architectural foundations that underlie the modulation of exemplarclassesfromthevoltage?gatedionchannel(VGIC)superfamilyandseektoaddressthefundamental question of how conformational changes in channel intracellular domains control and shape VGIC function. Many VGIC superfamily members, including Kv7 voltage?gated potassium channels and BacNaV bacterial voltage?gatedsodiumchannels,shareacommoncytoplasmicdomainarchitectureinwhichtheporedomain and a four?stranded coiled?coil frame a metastable membrane proximal domain that acts as a receiver for modulatory signals. We aim to understand how such metastable domains sense inputs from the calcium sensor calmodulin in Kv7s and from temperature in BacNaVs and transmit signals to the channel pore. The prevalence of similar intracellular elements among diverse VGICs suggests that the principles derived from thesestudieswillhavebroadimpactindefininghowsuchintracellularmodulesshapechannelresponses.A secondeffortisdirectedatdefiningthearchitectureofaclassofintracellularendolysosomalVGICsknownas Two?Pore?Channels(TPCs)andthathavelimitedstructuralcharacterization.Thesechannelspossessaunique tandem transmembrane architecture and respond to a variety of intracellular signals, including calcium. Elaboration of the underlying structural framework of exemplar VGICs is essential for understanding how theseandotherVGICsareintegratedintointracellularsignalingpathwaysandfordevelopingnovelwaysto intervene to control channel function. Our efforts encompass a multidisciplinary approach that includes biochemical, biophysical, X?ray crystallographic, and cryo?electronmicroscopy studies to probe structure and electrophysiological measurements to dissect function. Because of their important role in human physiology, VGICs are the targets for drugs with great utility for the treatment of cardiac arrhythmias, hypertension, congestiveheartfailure,epilepsy,andchronicpain.Thus,understandingtheirstructuresandmechanismsof action at atomic level detail should greatly assist the development of valuable therapeutic agents for a wide rangeofhumanailments.